U.S. patent application number 15/988421 was filed with the patent office on 2018-12-06 for cytotoxicity test method for medical devices.
The applicant listed for this patent is Novartis AG. Invention is credited to Xuemei Liu, Denise P. Rodeheaver, Robert Edward Rose, Stephen Paul Shannon, Charion Tolliver, Jamie Michaelis Walker, Jeffrey Charles White, Ann Marie Wright, Fan Zhang.
Application Number | 20180348205 15/988421 |
Document ID | / |
Family ID | 64459527 |
Filed Date | 2018-12-06 |
United States Patent
Application |
20180348205 |
Kind Code |
A1 |
Liu; Xuemei ; et
al. |
December 6, 2018 |
Cytotoxicity Test Method for Medical Devices
Abstract
A cytotoxicity method for medical devices that has equivalent
sensitivity, and greater efficiency compared to the colony
formation assay (CFA) direct contact method is developed. The
quantitative, direct contact cytotoxicity test method uses Mouse
lymphoma TK or human lymphoblastoid TK6 suspension cells for
medical device safety assessment. The advantages of using the
suspension cell assay over adherent V79-4 cells is that this
approach is quantitative, automated, not susceptible to potential
mechanical damage imparted by the overlying test materials, and
avoids issues related to non-adherence of cells to the test
material which might impact the interpretation of test results.
Inventors: |
Liu; Xuemei; (Fort Worth,
TX) ; Zhang; Fan; (Keller, TX) ; Rodeheaver;
Denise P.; (Fort Worth, TX) ; Wright; Ann Marie;
(Roswell, GA) ; Tolliver; Charion; (Fort Worth,
TX) ; Walker; Jamie Michaelis; (Burleson, TX)
; Rose; Robert Edward; (North Richlands Hills, TX)
; Shannon; Stephen Paul; (Arlington, TX) ; White;
Jeffrey Charles; (Southlake, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Novartis AG |
Basel |
|
CH |
|
|
Family ID: |
64459527 |
Appl. No.: |
15/988421 |
Filed: |
May 24, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62510542 |
May 24, 2017 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 41/46 20130101;
G01N 33/5014 20130101; C12M 1/34 20130101; C12M 23/12 20130101;
C12M 41/36 20130101; C12N 5/0634 20130101; G01N 15/00 20130101 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Claims
1. A method of quantifying the cytotoxicity of a medical device,
the method comprising: a) providing a medical device; b) incubating
the medical device with suspension cells in culture medium; c)
taking the images of the cells, d) determining the quantity of
living cells using a cell counter with image analysis.
2. The method of claim 1, wherein the suspension cells are Mouse
lymphoma TK or human lymphoblastoid TK6 cells.
3. The method of claim 2, wherein the suspension cells are Mouse
lymphoma TK cells.
4. The method of claim 1, wherein the culture of medium is select
from a group of heat-deactivated horse serum, sodium pyruvate,
pluronic F68 and combination thereof.
5. The method of claim 1, wherein the step of d) determining the
quantity of living cells is to use a cell counter with image
analysis cells based on the cell size, circularity, brightness, or
shape.
6. The method of claim 1, further comprising the steps of labeling
the cells with a dye.
7. The method of claim 6, further comprising the steps of detecting
the labelled cells.
8. The method of claim 6, wherein the dye is Trypan Blue.
9. The method of claim 1, wherein the incubating in step (b) is
effected in a multi-well plate.
10. The method of claim 7, wherein the incubating in step (b) is
effected in a 6, 24 or 96-multi-well plate.
11. The method of claim 1, wherein the medical device is a contact
lens.
12. The method of claim 1, wherein the medical device is a
stent.
13. The method of claim 1, wherein the medical device is a
scaffold.
14. The method of claim 1, wherein the medical device is an
implant.
15. The method of claim 1, wherein the cell counter is Vi-Cell XR
cell viability analyzer
Description
[0001] The invention relates to a method of using the suspension
cells to quantitatively measure cytotoxicity for a medical device,
such as a contact lens.
BACKGROUND OF THE INVENTION
[0002] The cytotoxicity testing is a primary requirement of all
major standards for medical devices. It allows rapid evaluation,
employs standard protocols, produces comparable data, and, due to
its sensitivity, enables potentially toxic materials to be
identified prior to in vivo testing. The high sensitivity of in
vitro cytotoxicity tests compared to animal studies might be due to
the direct exposure of cells to the material being tested and the
absence of the protective mechanisms that assist cells in vivo.
There are numerous screening cytotoxicity assays, whereas
international health authorities and standard organizations focus
on a more limited core test battery. Current recommendations or
guidelines for medical device submission vary from quantitative
(MTT/XTT, NR, and CFA) to qualitative assays (such as agar
diffusion, direct contact, and elution test). With the continuous
development in science and technology, new assays have evolved from
qualitative to quantitative, thus one or more quantitative
cytotoxicity assays are typically incorporated into the testing
paradigm during the development of new devices.
[0003] Generally, three types of quantitative cytotoxicity assays
are outlined in regulatory standards when evaluating materials for
medical devices: (1) tetrazolium reduction (MTT/XTT); (2) neutral
red (NR); and (3) colony formation (CFA). The MTT/XTT assay
requires metabolically viable cells to convert a substrate to a
colored product, whereas the NR assay is based on the lysosome
membrane integrity of healthy cells. Both methods evaluate cell
function using an automated UV/VIS plate reader to quantify the
signals of colored products or dyes. The signal changes are a
consequence of the adverse effects, which lead to dying cells
rapidly losing the basal cell function (i.e., mitochondrial
function, plasma membrane integrity). The cytotoxic effect is
indicated by significant changes of cell functional activities
compared with untreated controls, and thus indirectly measures the
cell number. In contrast, the CFA monitors a cell's ability to
produce a viable colony after treatment. Unlike the cell function
assays (MTT/XTT and NR), the CFA is unbiased to the mode of cell
death, since it directly measures the cytotoxic effect of a test
material, regardless of mechanism, as long as the test material
affects the cell's reproductive ability to form progenies. However,
the CFA method using Chinese hamster lung fibroblast (V79) cells
can be time consuming and because it is not automated, consistent
objectivity is difficult to achieve when counting colonies manually
under microscope.
[0004] One major difference between the two regulatory standards is
that the quantitative method outlined in International Organization
for Standardization (ISO) 10993-5 uses device extracts, while the
Japan Ministry of Health, Labor and Welfare (JMHLW) guideline
recommends using direct contact for devices whose leachables may be
inactivated during the extraction process and for devices that come
into direct contact with ocular tissues. In the CFA direct contact
method, cells are either cultured on the surface of the medical
device or, alternatively, the test material is placed directly on
sparsely populated cultured cells. For devices on which adherent
cells do not grow, such as contact lenses, the alternative method
is required. In contrast, a different direct contact assay outlined
in ISO, the agar diffusion/overlay, places the test material on a
thin layer of agar overlaying mammalian cell monolayer. Each direct
contact method has drawbacks. The disadvantages of using the agar
layer are that it is a qualitative method, and potentially
cytotoxic leachates may not be able to diffuse across the agar to
fully expose the cultured cells. Although the direct contact CFA
method is quantitative, the cells are more susceptible to potential
mechanical damage by the overlying test materials. In addition, the
measurement of the colony size is not included in the criteria for
cytotoxicity evaluation in the ISO and JMHLW standards. For this
reason, the direct contact CFA may not adequately capture the
adverse effect of a test material exhibited by colonies that are
smaller compared to control colonies but meet the test criteria of
>50 cells/colony. Thus, there continues to be an important need
for an automated quantitative method to assess cytotoxicity from
direct contact with a test material using equally sensitive cell
lines.
[0005] Due to the increased new types of medical devices to be
evaluated, there is still need for automated, high-throughput
screens that quantitatively assess cytotoxicity.
SUMMARY OF THE INVENTION
[0006] This invention is directed to a method of quantifying the
cytotoxicity of a medical device, the method comprising: a)
providing a medical device; b) incubating the medical device with
suspension cells in culture medium; c) taking the images of the
cells, d) determining the quantity of living cells using a cell
counter with image analysis.
BRIEF DESCRIPTION OF THE FIGURES
[0007] FIG. 1: Vi-Cell XR instrument parameter optimization. Mouse
lymphoma thymidine kinase (TK) cells were grown in RPMI.sub.10
medium to exponential phase. The cell numbers were quantified by
either the Vi-Cell XR or a hemocytometer. The data represent the
mean.+-.SD of three replicates performed at each dilution and the
correlation coefficient is indicated (R).
[0008] FIGS. 2A-2B: Mouse lymphoma TK cell growth rate. Cells were
seeded at 1.times.10.sup.4 cells/mL (.circle-solid. closed circle),
2.5.times.10.sup.4 cells/mL (.smallcircle. open circle), and
5.times.10.sup.4 cells/mL (.tangle-solidup. closed triangle), and
1.times.105 cells/mL (.DELTA. open triangle). A) Mouse lymphoma TK
cell growth rate using 5% fetal bovine serum (FBS). B) Mouse
lymphoma TK cell growth rate using 10% FBS. The data represent the
means from six replicates at each dilution, with the bars showing
standard deviation.
[0009] FIGS. 3A-3E: Direct contact colony formation assay. Fifty
V79 cells were seeded and allowed to attach to the bottom in each
well. Contact lenses were placed on top in close contact with the
V79 cells. On day 7 after seeding, contact lenses were removed and
cells were fixed and stained to determine colony formation rates.
A) Blank. B) Positive control--reference material B. C) Negative
control--wako disk. D) Senofilcon A contact lens. E) Polymacon
contact lens. The data represent six replicates.
[0010] FIGS. 4A-4B: Effect of benzalkonium chloride (BAK) and zinc
dibutyldithiocarbamate (ZDBC) on relative growth rate of mouse
lymphoma TK cells. A) Mouse lymphoma TK cells were treated with
serial dilutions of BAK for 72.+-.2 hours with either 5% FBS
(.circle-solid. closed circle) 10% FBS (.smallcircle. open circle).
B) Mouse lymphoma TK cells were treated a serial dilutions of ZDBC
for 72.+-.2 hours with either 5% FBS (.circle-solid. closed circle)
10% FBS (.smallcircle. open circle). The data represent the mean of
three replicates performed at each dilution, with the bars showing
standard deviation.
[0011] FIGS. 5A-5B: Effect of extracts of the reference material A
and B on relative growth rate of mouse lymphoma TK cells. Mouse
lymphoma TK cells were treated with different concentrations of
reference material A and B for 72.+-.2 hours. A) Reference material
A (5% FBS .circle-solid. closed circle; 10% FBS .smallcircle. open
circle). B) Reference material B (5% FBS .circle-solid. closed
circle; 10% FBS .smallcircle. open circle). The data represent the
mean of three replicates performed at each dilution, with the bars
showing standard deviation.
DETAILED DESCRIPTION
[0012] The present invention may be understood more readily by
reference to the following detailed description of the invention
taken in connection with the accompanying drawing figures, which
form a part of this disclosure. It is to be understood that this
invention is not limited to the specific devices, methods,
conditions or parameters described and/or shown herein, and that
the terminology used herein is for the purpose of describing
particular embodiments by way of example only and is not intended
to be limiting of the claimed invention. Any and all patents and
other publications identified in this specification are
incorporated by reference as though fully set forth herein.
[0013] Also, as used in the specification including the appended
claims, the singular forms "a," "an," and "the" include the plural,
and reference to a particular numerical value includes at least
that particular value, unless the context clearly dictates
otherwise. Ranges may be expressed herein as from "about" or
"approximately" one particular value and/or to "about" or
"approximately" another particular value. When such a range is
expressed, another embodiment includes from the one particular
value and/or to the other particular value. Similarly, when values
are expressed as approximations, by use of the antecedent "about,"
it will be understood that the particular value forms another
embodiment.
[0014] "Cells" are the building blocks of life because they make up
all the tissues and parts of our bodies. Cells are herein used in
its broadest sense in the art and refers to a living body which is
a structural unit of tissue of a multicellular organism, is
surrounded by a membrane structure which isolates it from the
outside, has the capability of self-replicating, and has genetic
information and a mechanism for expressing it. Cells used herein
may be naturally-occurring cells or artificially modified cells
(e.g., fusion cells, genetically modified cells, etc.).
[0015] "Medical device" refers to a device that is introduced
temporarily or permanently into a mammal for the prophylaxis or
therapy of a medical condition. For example, medical device may be
a contact lens or these devices include any that are introduced
subcutaneously, percutaneously or surgically to rest within an
organ, tissue or lumen.
[0016] Medical devices may include stents, covered stents such as
those covered with polytetrafluoroethylene (PTFE), or expanded
polytetrafluoroethylene (ePTFE), synthetic grafts, artificial heart
valves, artificial hearts and fixtures to connect the prosthetic
organ to the vascular circulation, venous valves, abdominal aortic
aneurysm (AAA) grafts, inferior venal caval filters, permanent drug
infusion catheters, embolic coils, embolic materials used in
vascular embolization (e.g., PVA foams), and vascular sutures.
Medical devices may include scaffolds. Scaffolds are defined as
three-dimension porous solid biomaterials designed to perform some
or all of the following functions: (i) promote cell-biomaterial
interactions, cell adhesion, and ECM deposition, (ii) permit
sufficient transport of gases, nutrients, and regulatory factors to
allow cell survival, proliferation, and differentiation, (iii)
biodegrade at a controllable rate that approximates the rate of
tissue regeneration under the culture conditions of interest, and
(iv) provoke a minimal degree of inflammation or toxicity in vivo.
The developing scaffolds with the optimal characteristics, such as
their strength, rate of degradation, porosity, and microstructure,
as well as their shapes and sizes, are more readily and
reproducibly controlled in polymeric scaffolds. Polymer scaffolds
can provide mechanical strength, interconnected porosity and
surface area, varying surface chemistry, and unique geometries to
direct tissue regeneration.
[0017] "Contact Lens" refers to a structure that can be placed on
or within a wearer's eye. A contact lens can correct, improve, or
alter a user's eyesight, but that need not be the case. A contact
lens can be of any appropriate material known in the art or later
developed, and can be a soft lens, a hard lens, or a hybrid lens. A
"silicone hydrogel contact lens" refers to a contact lens
comprising a silicone hydrogel material.
[0018] A "hydrogel" refers to a polymeric material which can absorb
at least 10 percent by weight of water when it is fully hydrated. A
hydrogel material can be obtained by polymerization or
copolymerization of at least one hydrophilic monomer in the
presence of or in the absence of additional monomers and/or
macromers or by crosslinking of a prepolymer. A "silicone hydrogel"
refers to a hydrogel obtained by copolymerization of a
polymerizable composition comprising at least one
silicone-containing vinylic monomer or at least one
silicone-containing macromer or a silicone-containing
prepolymer.
[0019] "Adherent cells", also called anchorage-dependent cells, are
grown in cell culture medium while attached to the bottom of a
tissue culture flask or other surface. Commonly, cells that come
from tissue are considered to be adherent. When the cells are added
to a tissue culture flask filled with cell culture medium and
allowed to sit for about one day, they will begin to settle on the
bottom and spread out. As they spread out, the cells secure a firm
hold by adhering to the bottom surface of the flask.
[0020] "Suspension Cell", also called anchorage-independent cell,
refers to a cell that can be grown by floating in the cell culture
medium. When suspension cells are looked at under the microscope,
they resemble tiny dots moving around in the liquid solution. There
will be few or no clumps compared to in an adherent culture.
[0021] "Lymphoma TK Cells" refers to the mouse lymphoma TK assay
(MLA) or human lymphoblastoid TK6 cells (both are commercially
available from ATCC (American Type Culture Collection) which are
used as a part of an in vitro battery of tests designed to predict
risk assessment prior to in vivo testing. The test has the
potential to detect mutagenic and clastogenic events at the
thymidine kinase (tk) locus. cells.
[0022] "Marker" refers to biomarker as "a characteristic that is
objectively measured and evaluated as an indicator of normal
biological processes, pathogenic processes, or pharmacologic
responses to a therapeutic intervention.
[0023] "Cell counter" Cell counters are tools for counting live
and/or dead cells in a culture. Any researcher who works in a cell
culture hood needs some sort of cell counting solution, whether to
determine cell concentration prior to cell passage, or to assess
cell viability following drug treatment. Both manual and automated
cell counters are available. Manual cell counting is generally
accomplished using a hemacytometer, a glass slide etched with a
counting grid. Automated cell counters operate either via
electrical impedence ("Electrical Sensing Zone" method), direct
imaging (either on a disposable slide or in a flow chamber), or
flow cytometry. The most important variable to consider in
selecting a cell counting solution is the ability to assess cell
viability. Electrical impedence (the Coulter Counting approach)
generally cannot distinguish live from dead cells (except by size);
but imaging-based approaches can, by using trypan blue to stain
dead (but not live) cells, just as in classic hemacytometry.
[0024] The invention is generally directed to develop a new
cytotoxicity method for medical devices that has equivalent
sensitivity, and greater efficiency compared to the colony
formation assay (CFA) direct contact method. The new, quantitative,
direct contact cytotoxicity test method uses mouse lymphoma L5178Y
thymidine kinase+/- (TK) suspension cells for medical device safety
assessment. Mouse lymphoma L5178Y thymidine kinase+/- (TK)
suspension cells are commercially available from American Type
Culture Collection (ATCC) (Cat. # CRL-9518, ATCC, Manassas, Va.).
The advantages of using the TK suspension cell assay over adhesion
V79-4 cells is that this approach is quantitative, automated, not
susceptible to potential mechanical damage imparted by the
overlying test materials, and avoids issues related to
non-adherence of cells to the test material which might impact the
interpretation of test results. For the new method, assays were
carried out in 24-well microplates; positive controls, negative
controls, and six commercial contact lenses were used to assess the
relative cell growth rate. Mouse lymphoma TK cells were seeded on
top (concave side) of the lenses with a cell concentration of
2.times.10.sup.4 cells/mL. Cell growth rate after 72 hours was
determined using the Vi-Cell XR (Beckmann Coulter), and relative
cell growth rate was calculated.
[0025] This method avoids the physical trauma caused by
unintentional movement/pressure of the test material to the
adhesion cells. Also, suspension cells can be easily cultured on
the surface of the medical device, whereas the apparent colony
forming ability of adhesion cells is decreased for a test material
on which cells hardly adhere. Therefore the use of suspension mouse
lymphoma TK cells appears promising for quantitative direct contact
cytotoxicity studies.
[0026] This application provides a method of quantifying the
cytotoxicity of a medical device, the method comprising: a)
providing a medical device; b) incubating the medical device with
suspension cells in culture medium; c) taking the images of the
cells, d) determining the quantity of living cells using a cell
counter with image analysis.
Material and Methods
[0027] The international standard cytotoxicity test reference
materials A (polyurethane film containing 0.1% zinc
diethyldithiocarbamate), B (polyurethane film containing 0.25% zinc
dibutyldithiocarbamate), and C (high-density polyethylene sheet),
were purchased from Hatano Research Institute (Hatano Research
Institute, Food and Drug Safety Center, Hatano, Kanagawa, Japan).
Wako tissue culture plastic disks were acquired from Wako Chemicals
(diameter (.PHI.): 14 mm, Cat. #162-09311, Richmond, Va.). Contact
lenses representing six different commercial lens polymers were
obtained from ABB Optical Group (Coral Springs, Fla.). The Vi-Cell
assay kit (Cat. #383198) was purchased directly from Beckman
Coulter (Brea, Calif.). All testing materials were freshly prepared
prior to each experiment.
[0028] Mouse lymphoma TK cell culture medium. All the cell culture
reagents were purchased from Thermo Fisher Scientific (Grand
Island, N.Y.) unless stated otherwise. As previously described by
Melvyn Lloyd 2012, the RPMI1640 medium (Cat. #11875),
heat-deactivated horse serum (Cat. #26050), sodium pyruvate (Cat.
#11360), pluronic F68 (Cat. #24040), and antibiotics were used in
this study. The basic medium (designated RPMI.sub.0) consisted of
RPMI1640 medium supplemented with 200 .mu.g/mL sodium pyruvate, 0.5
mg/mL pluronic F68, 100 U/mL penicillin and 100 .mu.g/mL
streptomycin (Cat. #15140). Growth media (designated RPMI.sub.10)
was RPMI.sub.0 with 10% (v/v) heat-deactivated horse serum. The TK
cells assay medium was RPMI1640.sub.0 supplemented with either 10%
(v/v) fetal bovine serum (FBS, Cat. #26140) or 5% (v/v) FBS and
designated as RPMI.sub.FBS10 and RPMI.sub.FBS05 respectively.
[0029] Mouse Lymphoma TK Cell Culture and Maintenance
[0030] The mouse lymphoma L5178Y TK.sup.-/- clone (3.7.2C) was
obtained from American Type Culture Collection (ATCC) (Cat. #
CRL-9518, ATCC, Manassas, Va.) and expansively cultured to make
master stocks (within 30 passages). Master stocks were maintained
in liquid nitrogen at a density of 2.times.10.sup.6 cells/mL with
growth media containing 5% dimethylsulfoxide (DMSO) (Cat. # D2650,
Sigma-Aldrich, St. Louis, Mo.). They were confirmed as free from
mycoplasma by ATCC. After thawing, mouse lymphoma TK cells were
grown at 37.degree. C. with a humidified atmosphere of 5% (v/v)
carbon dioxide (CO.sub.2) in air to achieve logarithmic growth. The
cells were routinely diluted to .about.2.times.10.sup.5 cells/mL
confirmed by either a hemocytometer or Vi-Cell XR to prevent
overgrowth (>2.times.10.sup.6 cells/mL) in growth media and were
used after two weeks of passage and during logarithmic growth.
[0031] Data Analysis
[0032] For each test material, data were collected and the mean and
SD were calculated using Excel. The IC.sub.50 was calculated using
SigmaPlot. Determination of cytotoxicity and test acceptance
criteria followed those described in JMHLW and ISO 10993-5: The
relative colony-forming rate/relative growth rate of positive
control (reference material B) should be equal or less than 10%,
and negative control (Wako disk) should be equal or greater than
80%. A test material was considered cytotoxic in the assay if the
relative colony formation rate/relative grow rate is less than 30%
as compared to the blank control and was considered negative if it
did not satisfy the above mentioned criteria.
Example 1: Vi-Cell XR Instrument Parameter Optimization
[0033] The Beckman Coulter Vi-Cell XR (Beckman Coulter,
Indianapolis, Ind.) is an automatic cell counter combined with
Trypan Blue dye as a marker of cytotoxicity, which penetrates the
cell membranes of dead cells with a characteristic blue color and
leads to easy differentiation of live (no blue color) and dead
(blue) cells. Using the Vi-Cell XR with video capture technology
(Vi-Cell XR Cell Viability Analyzer Software version 2.03), a cell
sample was taken and delivered to the flow cell for imaging. Cells
vary in their optical characteristics; therefore the optical
settings are important to correctly identify and quantify viable
versus non-viable cells. In this assay, the Vi-Cell XR was set to
capture 100 images per sample with the maximum cell size set as 20
micron and the minimum cell size set as 6 micron to exclude
cellular debris. Three cycles of both the aspirate and Trypan Blue
mixing were used to keep a single cell suspension and achieve good
mixing. The cell brightness (70%) and sharpness (100) were
optimized to determine whether the boundary "dark" pixels belonged
to a cell or were part of the background. The viable cell spot
brightness (75%) and area (5%) were used to determine whether a
cell was viable or non-viable. Minimum circularity was set as 0.8
to reject debris that exceeded the minimum cell diameter but was
too irregularly shaped to be treated as a viable cell. Validation
of the parameter optimization is described as follows:
[0034] An important criterion for developing sensitive and reliable
in vitro assays is the proper instrument parameter set up. To
confirm the optimization of the Vi-Cell XR parameters, we performed
a serial dilution of TK cells (2.times.10.sup.6, 1.times.10.sup.6,
5.times.10.sup.6, 1.times.10.sup.6, 5.times.10.sup.4, and
1.times.10.sup.4 cells/mL) and then the cell density was determined
with either a hemacytometer or the Vi-Cell cell counter. The
results (FIG. 1) showed that the readings from both the Vi-Cell XR
and hemacytometer increased linearly with increasing numbers of
cells, and the correlation of the results from hemocytometer and
Vi-Cell XR was very strong (R=0.999). Thus, the parameters for
Vi-Cell XR were optimized to accurately quantify TK cell numbers at
range of 1.times.10.sup.4 to 2.times.10.sup.6 cells/mL.
Example 2: Effect of Contact Lenses on Growth of Mouse Lymphoma TK
Cells in the TK Suspension Cell Assay
[0035] Assays were carried out in 24-well microplates (Cat. #3526,
Corning, Corning, N.Y.). To mimic real life situations, each
contact lens was removed from the saline in the manufacturer's
package and placed without blotting directly into each well of the
24-well plate containing 500 .mu.L RPMI.sub.FBS10 assay media. TK
cells were collected and reseeded (500 .mu.L, 4.times.10.sup.4
cells/mL) on the concave surface of the lens with a cell
concentration of 2.times.10.sup.4 cells/mL. Non-treated cells
served as the blank control, reference material B (diameter
(.PHI.): 14 mm) was the positive control, and Wako disk was the
negative control. After 72.+-.2 hours of incubation, 600 .mu.L
cells were mixed and transferred into the cell counter vial. The
cell growth rate after 72 hours was determined using the Vi-Cell
XR, and relative cell growth rate was calculated using the
following equation:
Relative cell growth rate (%)=[100.times.(Test cell concentration
at time 72 hours-mean of blank cell concentration at time 0)/(mean
of blank cell concentration at time 72 hours-mean of blank cell
concentration at time 0)]. The results are expressed as the
mean.+-.standard deviation (SD) from three independent studies and
each study has six replicates for each lens.
[0036] In addition to instrument parameter optimization, another
important criterion for cell growth inhibition assays is that the
cells should be at the logarithmic growth phase during the
treatment. The TK cell numbers were quantified by Vi-Cell XR,
serial dilutions were prepared, and seeding concentrations between
1.times.10.sup.4 and 1.times.105 cells/mL were used per assay with
six replicates. Then the TK cells were harvested and quantified at
0, 24, 48, 72, and 96 hours to generate the cell growth curve.
Since the doubling time for TK cells is 9-11 hours, the 72-hour
incubation time was chosen for the final assay so that the TK cells
could undergo at least 6 divisions. This is consistent with the CFA
using V79 cells, in which a colony is defined to consist of at
least 50 cells, which are formed after about 6 cell divisions. Our
data showed that both 1.times.10.sup.4 cells/mL and
2.5.times.10.sup.4 cells/mL TK cells were grown to exponential
phase for 72 hours incubation (FIGS. 2A and B). The TK cell number
at 1.times.105 cells/mL was significantly decreased on day 4 at 5%
FBS, which might be due to the deficiency of nutrient as compared
to the 10% FBS. The standard error between replicates was slightly
higher at 1.times.10.sup.4 cells/mL at earlier time points, which
might be due to the cell concentration being below the Vi-Cell XR
detection limit (5.times.10.sup.4 cells/mL) (data not shown).
Therefore, a seeding concentration at 2.times.10.sup.4 cells/mL was
employed for the studies. Since most of the historical data for
positive and negative controls in CFAs used FBS, the mouse lymphoma
TK cell (2.times.10.sup.4 cells/mL) growth rates were further
tested using different kinds of serum (Table 1). Our data showed
that FBS and horse serum had a similar impact on TK cell growth
rate.
TABLE-US-00001 TABLE 1 Mouse Lymphoma TK Cells Growth Rates Using
Different Kinds of Serum Cell Concentrations (.times.10{circumflex
over ( )}5 cells/mL).sup.a TK Growth Medium Time 0 Time 72 hours 5%
Horse Serum RPMI1640 0.19 .+-. 0.04 16.62 .+-. 0.87 5% Fetal Bovine
Serum RPMI1640 0.24 .+-. 0.05 14.92 .+-. 0.44 10% Horse Serum
RPMI1640 0.18 .+-. 0.02 18.70 .+-. 1.38 10% Fetal Bovine Serum
RPMI1640 0.23 .+-. 0.02 18.48 .+-. 0.70 .sup.aData represent the
mean .+-. SD from six replicates for each sample.
Example 3: Effect of Contact Lenses on Colony Formation of V79
Cells in the CFA Direct Contact Method
[0037] Testing was performed according to the JMHLW standard.
Chinese hamster lung cells, V79-4 (Cat. # CCL-93, ATCC, Manassas,
Va.) were propagated and maintained in M10 (single-strength Eagle's
Minimum Essential Medium with Earle's balanced salt solution (Cat.
# SH30024.01, HyClone, Logan, Utah) supplemented with 10% certified
FBS at 37.0.degree. C. in a gaseous environment of 5.0% CO.sub.2.
Briefly, V79 cell suspension after trypsinization was diluted to
100 cells/mL and then 500 .mu.L of cell suspension was added into
the 24-well plate containing 500 .mu.L of medium (M10). The culture
was incubated overnight at 37.degree. C., allowing the cells to
attach to the base of the plate. After incubation, the contact
lenses were added directly into each well (concave up). Non-treated
cells served as blank control, reference material B (diameter
(.PHI.): 14 mm) was the positive control, and Wako tissue culture
plastic disk was the negative control. After an additional 6 days
of culture, contact lenses were carefully removed; the cells were
fixed with methanol (Cat. # BDH20864, VWR, Suwanee, Ga.) and
stained with 2% Giemsa solution (Cat. #10092, ThermoFisher
Scientific, Grand Island, N.Y.). The number of colonies with 50
cells or more was manually counted using a Vista Vision microscope
(2.times.). The results are expressed as the mean.+-.SD based on
six replicates per lens.
[0038] Both positive control (reference material B) and negative
control (Wako disk) using the TK suspension cell assay showed
similar results as compared to the Japan direct contact CFA (Table
3). In addition, our data demonstrated an 83.0% correlation between
the TK suspension cell assay and the CFA direct contact method in
five of six commercial contact lenses studied. Although the
difference in incubation time might raise concerns about
consistency with the CFA results, our data demonstrate that those
theoretical concerns do not raise any practical issues with this
assay. Senofilcon A lenses showed a higher colony formation rate
(79.0.+-.9.8%) in the CFA direct contact assay compared to the cell
growth rate (17.1.+-.4.6%) using the mouse TK cells. This
inconsistency might be due to the fact that colony sizes treated
with senofilcon A were much smaller as compared to the blank
control, which might be an indication of a cytotoxicity effect
(FIG. 2). Since there is no cytotoxicity evaluation criterion for
colony size in the CFA, the mouse lymphoma TK cell growth
inhibition assay might be a more sensitive approach due to its
measurement of the total viable cells.
TABLE-US-00002 TABLE 2 Results of Direct Contact Assay Using Mouse
Lymphoma TK Suspension Cell Assay Versus CFA with V79 Cells. Mouse
Lymphoma TK CFA with V79 Cells Relative growth rate Relative colony
formation rate compared to blank (%).sup.b (%) - direct contact
method.sup.b Test articles (Mean .+-. STD) (Mean .+-. STD)
Reference Materials Positive - -0.5 .+-. 0.7 0 .+-. 0 reference
material B Negative - 89.4 .+-. 9.5 94.4 .+-. 10.9 Wako Contact
Lenses.sup.a Etafilcon A 10.3 .+-. 2.4 29.6 .+-. 9.6 Galyfilcon A
13.9 .+-. 1.4 18.1 .+-. 8.7 Senofilcon A 17.1 .+-. 4.6 79.0 .+-.
9.8.sup.c Balafilcon A 2.3 .+-. 3.1 3.5 .+-. 8.5 Methafilcon A 89.9
.+-. 11.1 91.3 .+-. 12.8 Polymacon 61.2 .+-. 18.5 83.5 .+-. 10.9
.sup.aContact lenses were removed from the package saline and used
without blotting. .sup.bData represent the mean .+-. SD from three
independent studies and each study has six replicates for each
lens. .sup.cSmaller colony size compared to blank.
Example 4: Effect of Benzalkonium Chloride (BAK) and ZDBC on Growth
of Mouse Lymphoma TK Cells in the TK Suspension Cell Assay
[0039] Phosphate buffer (Cat. # SH30028.02, HyClone, Logan, Utah)
was used for dilutions of BAK (1.25, 2.5, 5, 10, 25, and 50 parts
per million) (Cat. # B1068, Spectrum Chemical, New Brunswick,
N.J.). A total of 900 .mu.L of 2.2.times.10.sup.4 cells/mL TK cells
in assay medium (RPMI.sub.FBS10 or RPMI.sub.FBS05) were seeded in a
24-well plate and then treated with different dilutions of BAK (100
.mu.L) to achieve final concentrations of 0.125, 0.25, 0.5, 1.0,
2.5, and 5 ppm. Phosphate buffer (100 .mu.L)-treated cells served
as a blank control, reference material B (diameter (.PHI.): 14 mm)
as a positive control, and Wako tissue culture plastic disk served
a negative control. After 72.+-.2 hours incubation, 600 .mu.L cells
were mixed and transferred into the counter vial. The relative cell
growth rate was determined and calculated as described above. The
results are expressed as the mean.+-.SD based on three
replicates.
[0040] DMSO was used to dissolve ZDBC powder (Cat. # Z1031,
Spectrum Chemical, New Brunswick, N.J.) to make 0.05, 0.1, 0.2,
0.4, 0.8, and 2 mg/mL dilutions. Next, 995 .mu.L of
2.0.times.10.sup.4 cells/mL TK cells in assay medium (either
RPMI.sub.FBS10 or RPMI.sub.FBS05) were seeded in a 24-well plate
and treated with 5 .mu.L of different ZDBC dilutions to achieve
final concentrations of 0.25, 0.5, 1, 2, 4, and 10 .mu.g/mL. DMSO
(5 .mu.L)-treated cells served as a blank control, reference
material B (diameter (.PHI.): 14 mm) as a positive control, and
Wako tissue culture plastic disk served a negative control. After
72.+-.2 hours incubation, 600 .mu.L cells were mixed and
transferred into the counter vial. The relative cell growth rate
was determined and calculated as described above. The results are
expressed as the mean.+-.SD based on three replicates.
[0041] BAK and ZDBC both showed a dose-dependent decrease in cell
growth. BAK at 5, 2.5, 1, and 0.5 ppm yielded significant cell
growth inhibition as compared to the blank control. Both 0.25 and
0.125 ppm BAK were not cytotoxic (FIG. 4A). The 5% FBS (BAK:
IC.sub.50=0.55 ppm) and 10% FBS (BAK: IC.sub.50=0.59 ppm) did not
have any impact on cytotoxicity for BAK treatment. ZDBC showed
lower IC.sub.50 using the 5% FBS medium (IC.sub.50=2.0 .mu.g/mL) as
compared to the 10% FBS medium (IC.sub.50=4.5 .mu.g/mL) (FIG. 4B),
which is consistent with the reference results in the JMHLW
standard.
Example 5: Effect of Reference Material Extracts on Growth of Mouse
Lymphoma TK Cells in the TK Suspension Cell Assay
[0042] Assays were carried out in 6-well plates (Cat#3506, Corning,
Corning, N.Y.). Each extraction was prepared according to ISO
10993-12 (2012) and JMHLW standards. Briefly, reference materials
A, B, and C were extracted (reference materials A and B, 0.1 g/mL;
reference material C, 0.2 g/mL) using assay medium (RPMI.sub.FBS10
or RPMI.sub.FBS05) for 24 hours at 37.degree. C. Each extraction of
reference materials was further diluted using assay medium
(RPMI.sub.FBS10 or RPMI.sub.FBS05). Three (3) mL of reference
material A (2%, 1%, 0.5%, 0.25% and 0.125%), reference material B
(100%, 75%, 50%, 25%, and 12.5%), or reference material C (100%)
dilutions were added into wells of a 6-well plate. Cells were
collected and reseeded (30 .mu.L, 2.times.10.sup.6 cells/mL) into
each well. After 72.+-.2 hours incubation, 600 .mu.L cells were
mixed and transferred into the counter vial. The relative cell
growth rate was calculated as described above. The results are
expressed as the mean.+-.SD on three replicates.
[0043] The 100% extract of reference material C did not have any
impact on the relative cell growth rate (5% FBS: 89%.+-.10%; 10%
FBS 100%.+-.9%). The extract of reference material A was
significantly more cytotoxic than that of reference material B, and
both behaved in a dose-dependent manner (FIGS. 5A and 5B). In
addition, both reference material A (5% FBS: IC50=0.26%; 10% FBS:
IC50=0.49%) and reference material B (5% FBS: IC50=53%; 10% FBS:
IC50=78%) had lower IC.sub.50 using 5% FBS medium as compared to
10% FBS medium, which is also consistent with the reference results
in the JMHLW standard.
DISCUSSION AND CONCLUSIONS
[0044] Cytotoxicity testing is an important endpoint for biological
safety evaluations of medical devices and provides a standardized,
sensitive, and inexpensive means to determine whether a test
material contains significant quantities of potentially
biologically harmful leachables. The results obtained from
cytotoxicity test systems have indicated that the relative toxicity
can vary not only according to exposure time, cultured cell type,
and presence or absence of serum, but also by the choice of
endpoints. There are a variety of assay technologies ranging from
qualitative to quantitative that utilize the device or its extracts
as the test material to estimate the cell viability following
direct contact. Each cell viability assay has its own set of
advantages and disadvantages (Table 3). Regardless of the assay
method chosen, the major critical factors for reproducibility and
accuracy include: (1) using a tightly controlled, well
characterized, and consistent source of cells, and (2) performing
appropriate characterization of reagent concentration and
incubation time for each experimental model system.
TABLE-US-00003 TABLE 3 Comparison of Mouse Lymphoma TK and Current
Quantitative Cytotoxicity Testing Method Assays TK Suspension Cell
Assay JMHLW Colony Formation Direct Contact Cell line L5178Y
thymidine kinase+/- V79 (TK) Cell type Mouse Lymphoma Chinese
hamster lung fibroblast cells Cell Well characterized Well
characterized adhesion cells characteristics suspension cells
Treatment time 72 hours 6-7 days Measurement Quantitative/Number of
viable Quantitative/Number of colonies cells Assay Cell growth Cell
growth mechanism Instrument Cell Counter Manually count Automation
Yes No Test material Direct contact and medical Direct contact and
medical device device extract. extract. Method criteria Direct: A
test material is Direct: The test material is judged considered
cytotoxic in the as cytotoxic when the IC.sub.50 is 100% assay if
the relative grow rate or lower in the extraction method is less
than 30% as and the colony formation rate of compared to the blank
the direct contact method is less control. than 30%. Extract: The
test material is Extract: The test material is judged as cytotoxic
if the judged as cytotoxic when the relative grow rate in the 100%
colony formation rate in the 100% test solution shows a test
solution shows a decrease decrease beyond 30% of the beyond 30% of
the rate in the rate in the blank control blank control group (the
colony group. formation rate <70%). Advantages Quantitative,
automated, and Quantitative, direct measurement not susceptible to
potential of cell growth. mechanical damage imparted More historic
data by the overlying test No special equipment needed materials,
and avoids issues related to non-adherence of cells to the test
material. Disadvantages Special equipment needed Labor intensive;
potential Need further validation and mechanical damage by
overlying characterization test materials, and adhesion cells might
not grow on the surface of the medical device. Only one side of the
material can be evaluated. Assays ISO - Agar Overlay ISO - Direct
Contact Cell line L-929 L-929 Cell type Mouse fibroblastic cell
Mouse fibroblastic cell Cell Well characterized adhesion Well
characterized adhesion characteristics cells cells Treatment time
24 hours 24 hours Measurement Qualitative Qualitative Assay
Cytotoxicity Cytotoxicity mechanism Instrument Microscope
Microscope Automation No No Test material Indirect contact with
test Direct contact material Method criteria The biological
reactivity is The biological reactivity is rated rated on a scale
of 0-4. The on a scale of 0-4. The test test material is judged as
not material is judged as not cytotoxic if the response is
cytotoxic if the response is not not greater than grade 2. greater
than grade 2. Advantages Avoid mechanical damage by Direct contact
with cells. overlying test materials. More historic data More
historic data No special equipment needed No special equipment
needed Disadvantages Qualitative and cytotoxic Qualitative and
potential leachates may not be able to mechanical damage by
overlying diffuse across the agar to fully test materials. Only one
side of expose the cultured cells. the material can be evaluated.
Only one side of the material can be evaluated.
[0045] Although MTT/XTT and NR assays are used extensively as
convenient, sensitive, and rapid measures of cell viability, each
of the method has their disadvantages and must be used with
caution. For example, reducing agents and respiratory chain
inhibitors could potentially affect the MTT formazan formation of
mitochondrial MTT reduction in the MTT/XTT assay. In terms of the
NR assay, an increase in NR uptake was demonstrated to have been
induced by lysosomal swelling agents such as weakly basic
substances and by osmotic swelling agents such as polyols. The
lysosomal swelling may lead to an underestimation of the
cytotoxicity when the NR assay is used. The mouse lymphoma TK
suspension cell assay we described here directly monitors a cell's
ability to propagate after treatment using the video capture
imaging technology. Our goal was to develop a cell proliferation
assay with direct contact to a medical device using automated
technology, thereby increasing efficiency in performing
large-scale, reproducible experiments. This new method uses an
automatic cell counter combined with Trypan Blue dye as a marker of
cytotoxicity, which penetrates the cell membranes of dead cells
with a characteristic blue color and leads to easy differentiation
of live (no color) and dead (blue) cells. While there are a number
of cell lines that can be used, the mouse lymphoma TK cell line is
preferred based on a body of research that exists for this cell
line. It is commonly used in a standard in vitro mammalian gene
mutation assay and has the ability to grow in suspension. Chinese
hamster lung (V79) and ovary (CHO) cells, used in CFAs, were also
used in in vitro mammalian gene mutation assays use. The
quantitative cell function assays (MTT/XTT and NR) are using
biochemical methods to indirectly measure the inhibition of cell
growth. The effects on cell function are a consequence of
non-specific alternations in "basic cell functions" (i.e.,
mitochondria, plasma membrane integrity, etc.), which may then lead
to impairment or death of the cell. Since these are early cellular
changes, the lethality or reversibility of the effect may be
unknown. The new TK suspension cell assay is unbiased to the mode
of cell death and is able to detect the cytotoxic effect of a test
material, regardless of mechanism. Another advantage of this
approach is the ability to seed TK cells on top of the test
materials, which achieves direct contact without physical trauma to
seeded cells and avoids confounding effects when cells are not able
to adhere to the test material. Cell density and cytotoxicity are
easily determined using a cell counter combined with the Trypan
Blue stain. Furthermore, this TK method is more sensitive as
compared to the CFA direct contact which reports only colony number
and does not include the colony size in the criteria for
cytotoxicity evaluation as long as there are more than 50 cells per
colony. This feature may mask the adverse effect exhibited by
smaller colonies in test materials.
[0046] In summary, data demonstrate that this TK suspension cell
assay using the L5178 TK.sup.+/--3.7.2.C mouse lymphoma cell line
performs equivalently in determination of potential cell growth
inhibition to the direct contact colony formation assay described
in several regulatory standards. This method represents a valuable
addition to the battery of assays for detection of potentially
toxic materials leaching from a test material that is either a
solid or liquid. Further work would be required to focus on
large-scale and formal validation, which would provide more
information about the criteria applied in this assay and prove its
applicability for medical device safety assessment.
[0047] The invention has been described with the aid of a specific
embodiment of the process or apparatus, respectively. However, the
invention is not limited to the specific embodiment described but
rather various changes and modifications are possible without
departing from the general concept underlying the invention.
Therefore, the scope of protection is defined by the appended
claims.
* * * * *